Project Summary Triple negative breast cancer (TNBC) is a subtype of breast cancer that occurs in 15-20% of patients, and is defined by tumors that do not overexpress the estrogen, progesterone and HER2 receptors. This aggressive subtype has a significantly worse overall survival compared to non-TNBCs and importantly, these patients lack options for targeted therapy. To identify novel subtype-specific therapeutic targets, we must first understand the biological underpinnings of the disease. DNA methylation is a hallmark of cancer, as it can regulate gene expression of both tumor suppressor genes and oncogenes. We found TNBC tumors have genome-wide hypomethylation compared to other subtypes and normal breast controls. Our preliminary data suggest most of this hypomethylation occurs outside of CpG islands (CGI). TET1 is a DNA demethylase that converts methylated cytosine into 5-hydroxymethylcytosine, which can then be further oxidized or converted to un- methylated cytosine. TET1 has a CXXC DNA binding domain that allows it to recognize and bind to CGIs, but this domain limits its ability to demethylate sites outside of CGIs. However, we discovered a novel isoform of TET1 (TET1ALT) that lacks the CXXC domain, but retains the demethylase domain. Thus, TET1ALT may contribute to the demethylation observed outside of CGIs. Our analyses of TCGA data revealed TET1 and TET1ALT are upregulated in a subset of TNBC patients and that this is associated with hypomethylation and activation of genes in the PI3K pathway. We hypothesize that a subset of TNBC tumors upregulate TET1 and TET1ALT, leading to demethylation and activation of oncogenes, thereby driving tumorigenesis. We plan to test our hypothesis by overexpressing both TET1 and TET1ALT in normal mammary cells and measuring genome- wide DNA methylation, hydroxymethylation, and gene expression changes. This will allow us to determine if TET1, TET1ALT or both are leading to the demethylation and PI3K activation that we observed in our TCGA analyses. We will investigate whether the TET1 isoforms affect transformation in vitro and tumorigenesis in vivo by using xenograft and transgenic mouse models. This proposal will provide the mechanistic basis for targeting PI3K in breast cancer, provide methylation based biomarkers for response to therapy and further, will validate TET1 as a potential new druggable target that could be very specific for TNBC.